The goal of the Section on Human Genetics is to identify and study the function of mutated genes for human hereditary deafness. This work begins with the ascertainment of large families in which deafness appears to be inherited either as a dominant or a recessive trait. We then search for linkage of the deafness to genetic markers for the known (already reported) syndromic, DFNA (dominant) and DFNB (recessive) loci. If linkage to the known deafness loci can be excluded, we initiate a genome-wide screen to search for novel deafness loci followed by work to identify the causative gene. During the past year we ascertained several large families segregating deafness, mapped a novel deafness locus, and identified a novel gene for nonsyndromic deafness. Sections of the following projects were completed in the past year and have been published or are in press and likely to be published in 2013. 1. We will determine how noncoding mutations (del3 and del10 mutations) of HGF cause deafness DFNB39, which is the subject of ongoing work by Drs. Julie Schultz and Rob Morell. Hepatocyte growth factor is the 728 amino acid polypeptide product of the longest transcript arising from HGF (isoform 1). HGF activates downstream signaling only after proteolytic cleavage of the pro-HGFpolypeptide to form alpha and beta chains. HGF has indispensible functions involving growth and wound repair, and somatic mutations of HGF have been implicated in some cancers. There are also naturally occurring shorter isoforms of HGF that arise due to alternate splicing. Isoforms 3 and 4 use an alternate splice acceptor site in exon 5, resulting in the omission of five amino acids. Although not annotated as such in RefSeq, our own data demonstrates that isoform 5 also occurs in both an exon 5a and 5b configuration. In addition, we discovered a shorter HGF transcript designated isoform 6, which can be RT-PCR amplified from many tissues, including cochlea. The 3'-UTR of isoform 6 transcripts include the deleted sequence of DFNB39 mutations. Despite a wealth of research on HGF, no comprehensive study of the temporal and spatial regulation of HGF isoforms has been made. Previously we described two different Hgf mouse models and demonstrated that both have hearing loss (Schultz et al., 2009). An Hgf transgene ubiquitously over-expresses full length HGF, and a conditional knockout of Hgf in which exon 5 was flanked by flox sequences. Taken together, these data indicate that dysregulation (too much or too little) of HGF causes hearing loss. To recapitulate deafness due to the del3 and del10 mutations in the 3-UTR of human HGF isoform 6, we have engineered an orthologous del10 deletion of the 3-UTR of Hgf isoform 6 in mouse. This project promises to provide details of the necessary function of HGF in the auditory system. 2. We recently mapped a novel nonsyndromic deafness locus (DFNB81) to chromosome 19p, which is distinct from the closely linked DFNB72 locus (Rehman et al., 2011 EJHG). Mutations of GIPC3 are responsible for DFNB72 deafness. Using next-generation sequencing technology Atteeq rehman, PhD, a fellow in the LMG, identified mutations in CLPP encoding a mitochondrial chambered proteas. DFNB81 deafness was initially presumed to be nonsyndromic. However, further clinical chactareization of the affected subjects indicates Perrault syndrome characterized by hearing loss and female gonadal dysgenesis. This paper was published earlier in 2013 (Jenkinson, rehman et al., AJHG 2013). Atteeq is the co-first author and Friedman is the co-communicating author with William Newman, MD, PhD. Meghan Drummond, PhD, a fellow in the LMG/NIDCD is continuing to work on the function of CLPP in the auditory system. 3. In 2011 we mapped a novel locus for nonsyndromic deafness to chromosome 16p. The locus is designated DFNB86 (Ali et al., 2011). recently, Atteeq Rehman, a fellow in the LMG, identified mutations in a novel deafness gene. A publication is being prepared as a acollaboration with Suzanne Leal, PhD at baylor College of Medicine and Andrew J. Griffith, MD, PhD, NIDCD/NIH. 4. Grhl2-TMinsC/+ is a mouse model of DFNA28 human progressive hearing loss, which we reported ten years ago (Peters et al., 2002). A goal of our present study is to understand the function of the GRHL2 transcription factor in the auditory system. To that end, we have performed ChIP-Seq experiments using our own and commercially available antibodies to GRHL2, in a variety of tissues and cell types. In collaboration with Drs. Maria Ramirez and Saaket Varma at Boston University Medical School, and as a way to validate this technology in our laboratory, we extended our ChIP and expression analyses to include developing lung buds and the MLE15 cell line derived from adult mouse lung. GRHL2 is required for normal lung development. Varma et al. 2012, (Morell and Friedman are co-authors) showed that GRHL2 and NKX2-1 transactive each other and form a regulatory loop defining the critical transition from a type II cell type (cuboidal) to type I (squamous) in the lung alveolus. We are making a more comphrehensive survey of GRHL2 regulatory targets in the lung as compared to the auditory system using ChIP-Seq and our AB5500 sequencer. 5. Nonsyndromic deafness DFNB48 and a novel type 1 Usher syndrome were previously genetically mapped by us to an overlapping interval on chromosome 15q21-q23 (Ahmed et al., 2009). We recently demonstrated that mutations of CIB2 are associated with DFNB48 and USH1J are allelic and that the causative gene encodes a calcium binding protein (Riazuddin et al., November 2012, Nature Genetics). 6. Meghan Drummond PhD is working on the structure and dynamics of the gamma-actin and beta-actin in stereocilia using live-imaging and mouse mutants of gamma-actin some of which are equivalent to those alleles associated with dominantly inherited deafness DFNA20, a locus genetically mapped and reported by staff of the LMG several years ago and positionally cloned and identified as ACTG.